Diagnostic preparation for the identification of beta-hemolytic streptococci

ABSTRACT

A PREPARATION FOR THE BIOCHEMICAL IDENTIFICATION OF BHEMOLYTIC STREPTOCOCCI INTO SEROLOGIC GROUPS, E.G., GROUPS A, B, C, D, ETC., IS DISCLOSED. THIS PREPARATION COMPRISES A CARRIER, E.G., A BIBULOUS STRIP CONTAINING A GLYCEROL REAGENT BAND, A SALICIN REAGENT BAND AND AN ESCULIN REAGENT BAND. IN THE GLYCEROL AND SALICIN REAGENT BANDS THERE ARE ALSO PH INDICATOR SYSTEMS. THE ESULIN BAND CONTAINS A COMPOUND WHICH REACTS WITH ESULIN BREAKDOWN PRODUCTS TO PRODUCE A COLOR CHANGE. THESE BANDS ARE SEPARATED FROM EACH OTHER BY HYDROPHOBIC BARRIERS. IN USE, A QUANTITY OF THE BETA HEMOLYTIC STREPTOCOCCUS WHOSE SEROLOGICAL IDENTIFICATION IS TO BE DETERMINED IS TRANSFERRED ONTO EACH OF THE THREE REAGENT BANDS. A DROP OF SALINE OR WATER IS ADDED TO THE SALICIN AREA AND TO THE ESCULIN AREA. THE STRIP IS PLACED IN A TUBE CONTAINING 0.3 ML. OF SALINE OR WATER TO WET THE GLYCEROL BAND. THE REAGENT IMPREGNATED STRIP IS INCUBATED AT APPROXIMATELY 37*C. FOR TWO TO FOUR HOURS. SUGAR FERMENTATION IS MEASURED BY AN INDICATOR COLOR CHANGE AND ESCULIN HYDROLYSIS BY FORMATION OF A GREY-BLACK COLOR. GROUP A STREPTOCOCCI, WHICH CAUSE MOST OF THE CLINICAL MANIFESTATIONS SUCH AS RHEUMATIC FEVER AND NEPHRITIS, ARE ESCULIN NEGATIVE, GLYUCERIN NEGATIVE AND SALICIN POSITIVE. GROUP B AND C STREPTOCOCCI MAY BE INVOLVED IN DISEASES BUT MORE OFTEN ARE PART OF A NORMAL FLORA IN A PRIMARY ISOLATION BLOOD PLATE SHOWING B-HEMOLYSIS. IT IS IMPORTANT TO DIFFERENTIATE BETWEEN THESE GROUPS AND GROUP A. GROUP D STREPTOCOCCI, KNOWN AS ENTEROCOCCI, ARE OFTEN ASSOCIATED WITH URINARY TRACT INFECTION AND RAPID IDENTIFICATION OF THESE ORGANISMS IS ALSO CLINICALLY IMPORTANT.

mam AVAILABLE COPY Oct. 17, 1972 D. P. KRONISH ETAL 3,699,003

DIAGNOSTIC PREPARATION FOR THE IDENTIFICATION OF ,6 -HEMOLYTIC STREPTOCOCCI Filed July 13, 1970 AREA NUMBER INVENTORS DONALD P. KRONISH LEE S. ZURIFF United States Patent Office Patented Oct. 17, 1972 U.S. Cl. 195-103.5 R 8 Claims ABSTRACT OF THE DISCLOSURE A preparation for the biochemical identification of phemolytic streptococci into serologic groups, e.g., groups A, B, C, D, etc., is disclosed. This preparation comprises a carrier, e.g., a bibulous strip containing a glycerol reagent band, a salicin reagent band and an esculin reagent band. In the glycerol and salicin reagent bands there are also pH indicator systems. The esculin band contains a compound which reacts with esculin breakdown products to produce a color change. These bands are separated from each other by hydrophobic barriers. In use, a quantity of the beta hemolytic streptococcus whose serological identification is to be determined is transferred onto each of the three reagent bands. A drop of saline or water is added to the salicin area and to the esculin area. The strip is placed in a tube containing 0.3 ml. of saline or water to wet the glycerol band. The reagent impregnated strip is incubated at approximately 37 C. for two to four hours. Sugar fermentation is measured by an indicator color change and esculin hydrolysis by formation of a grey-black color. Group A streptococci, which cause most of the clinical manifestations such as rheumatic fever and nephritis, are esculin negative, glycerin negative and sa'licin positive. Group B and C streptococci may be involved in diseases but more often are part of a normal flora in a primary isolation blood plate showing fl-hemolysis. It is important to differentiate between these groups and Group A. Group D streptococci, known as enterococci, are often associated with urinary tract infection and rapid identification of these organisms is also clinically important.

Streptococci are gram positive organisms, spherical or oval in shape and have a tendency to grow in chains. Those streptococci which possess primary pathogenicity for man and animals belong to the subdivision of hemolytic streptococci. This classification is based on the observation that these streptococci cause lysis of red blood cells, a property which is useful in the initial identification of these organisms in culture material. In a blood plate showing a clear zone resulting from lysis of the red blood cells, the organism is defined as B-hemolytic. Other types of hemolysis have also been described.

As a result of extensive serological studies, B-hemolytic streptococci have been divided further into a number of distinctive serological groups, which are related in some degree to the natural habitat and the pathogenicity of the organism. Consequently, serological identification has become an important step in the identification of pathogenic streptococci. It is generally known that hemolytic streptococci belonging to the serological Groups A and D are not only responsible for the acute streptococcal diseases of man, but are also involved in other diseases such as rheumatic fever, nephritis, bacteriuria and others. Accordingly, when ,[i-ltemolytic streptococci are isolated, it is important that the clinician know the serological group to which the organism belongs so that appropriate treatment can be initiated. For example, when a physician detects fl-hemolytic streptococci in a culture from the upper respiratory tract and does not type the isolate, he will frequently administer an antibiotic for a prolonged period of time to prevent possible sequella. In many cases, where the organism is not a Group A streptococci antibiotic treatment is not necessary.

In the serological groups as described by Lancefield (Journal of Experimental Medicine 57: 571-595, 1933), identification is a function of the presence of specific groups of cellular antigens. These antigens are detected by precipitin reactions between solutions of extracts of an unknown streptococcus culture and antisera prepared by immunizing rabbits with heat-killed suspensions of streptococci. It is readily appreciated that such a technique is quite time-consuming, and requires highly trained personnel.

We have now found a method whereby fi-hemolytic streptococci can be readily identified into serological groups by a rapid and positive biochemical method. The practice of this invention is more easily understood" by the reference to the accompanying drawing which is a diagrammatic representation of the invention.

Broadly speaking, the preparation of this invention is a carrier, typically a strip of a bibulous material which contains a plurality of distinct areas.

Area 1 is impregnated with glycerol, a pH indicator system, and optionally containing nutrient medium and having a pH on the alkaline side of the pH indicator used.

Area 2 is a hydrophobic barrier which is capable of preventing the migration of the ingredients. Any substance which will form a waterproof barrier can be advantageously employed. Suitable materials include, for example, waxes, lacquers and a colorless acrylic resin known as Krylon Crystal Clear: this Krylon material is particularly preferred. It is supplied in a hydrocarbon vehicle and may be diluted for ease of application with toluene or other hydrocarbon solvents, for example, methyl, ethyl or isopropyl alcohol.

Area 3 is impregnated with salicin, a pH indicator system and optionally containing a nutrient medium and having a pH on the alkaline side of the pH indicator used.

Area 4 is a hydrophobic barrier which is the same as previously described for area 2.

Area 5 is impregnated with esculin and a ferric salt, for example, ferric chloride, ferric ammonium citrate, ferric nitrate, ferric citrate, or an aluminum salt, for example, aluminum ammonium sulfate, or a lead salt for example, lead acetate or lead nitrate.

Area 6 is a hydrophobic barrier which is the same as previously described for area 2.

Area 7 is optional and can be impregnated with any hydrophobic material as previously described for area 2 and any appropriate dye serving as an identification means.

The sequence of reagent areas on the strip is not critical as long as each area is separated by a hydrophobic barrier but the configuration described is preferred.

The indicator system for areas -1 and 3 are those indicators which will give a color change between about pH 9 to about pH 5. Examples of these indicators are chlorophenol red, bromocresol purple, bromophenol red, bromothymol blue, neutral red, u-naphtholphthalein and phenol red. Examples of nutrient media are those which are traditionally used in bacterial culture, for example, peptone, tryptone, proteose, tryptose, yeast extract, etc.

Bibulous materials which can be employed as the carrier are, for example, those materials which by means of a capillary action can draw a liquid upwards; or materials such as filter paper, felt, porous, ceramic strips, woven or matted glass fiber and the like are suitable.

Alternatively, the active ingredients can be impregnated on any absorbent material and then mounted on a suitable support, for example, a plastic strip.

In use, a loopful of organism showing fl-hemolysis on a blood agar plate is rubbed into the bottom of area 1 and into areas 3 and 5. A drop of saline or water is also added to areas 3 and 5. The resulting system is then immersed in a 13 x 100 mm. or similar size test tube containing 0.3 ml. of saline and incubated at 37 C. for about 2 to 4 hours.

A positive reaction in areas 1 and 3 when phenol red is the indicator system is indicated by the development of a yellow color; a negative color is indicated by the persistence of a red color. The positive reaction is as a result of the fermentation of the sugars and the resulting acidity.

A positive reaction in area is indicated by the development of a gray-black color, whereas a negative reaction is indicated by the absence of color change. Again, a positive reaction is the result of the fermentation of esculin and the reaction of hydrolysis products with ferric salts or other color producing compounds.

By the observation of the presence or absence of the biochemical changes, the serological identification of B-hemolytic streptococci is readily obtained from the ollowing chart:

Streptococcus group A B C D G Esculin GlyceroL- Sallein 1* 1* 1* Nora-11* means variable.

EXAMPLE I Part A-Preparation of glycerol reagent solution Ingredients: For each 100 ml.

(1) Glycerol USP About -20 g. (2) Peptone About 1-2 g. (3) Phenol red 200 mg. (4) Distilled water qs. 100 ml. (5) 5.0N NaOH qs.

Dissolve (1), (2) and (3) in 90 ml. of (4) with constant stirring. Adjust the pH 11.0 with (5). Bring to volume with (4).

Part B-Preparation of salicin reagent solution Ingredients: For each 100 ml.

( 1) Salicin About 10-20 g. (2) Peptone About 1-2 g. (3) Phenol red 200 mg. (4) Distilled water qs. 100 ml. (5) 5.0N NaOH qs.

Heat 60 ml. of (4) to 70 0.; add (1), (2) and (3) and dissolve with constant stirring. Adjust pH to 10.0 with (5). Bring to volume with (4). Maintain 70 C. temperature.

Part CPreparation of esculin reagent solution Ingredients: For each 100 ml.

(1) Esculin 2 to 5 gm. (2) Ferric ammonium citrate 1 to 2.5 gm.

(3) Water qs.

(4) 1 N sodium hydroxide solution qs.

Heat 80 ml. of (3) to about 70 C. Add (1) and (2) and dissolve with constant stirring. Maintain temperature at 50 C.-70 C. Adjust to pH 4.3 with (4) at 70 C. Since the esculin-ferric ammonium citrate mixture is light sensitive, the preparation should preferably be protected from light.

Part D-Application to bibulous material In preparing the test strip, the initial step is to prepare the barrier areas 2 and 4 which act to prevent the migration of the reagent solutions which are subsequently supplied. These barrier zones are applied to suitable carrier, preferably Eaton-Dikeman No. 623 filter paper, by employing a solution of a lacquer which produces a water impervious barrier. After allowing the lacquer solution to dry, the glycerin reagent solution, the salicin reagent solution, and the esculin reagent solution prepared according to the above description is then applied to areas 1, 3 and 5. The areas thus formed are allowed to dry; the filter paper is then cut into strips.

EXAMPLE II The following are examples but not the only methods of use of reagent strips:

One or two loopfuls of streptococci showing ,B-hemolysis on a blood agar plate is rubbed on the bottom of area 1 and onto areas 3 and 5 respectively. The test system thus produced is added to a 13 x mm. tube containing 0.3 ml. of saline so that area 1 is immersed in the saline and incubated 2 to 4 hours at 37 C. The identification of the serological group to which the organism belongs is readily determined by reference to the aforementioned table.

We claim:

1. A diagnostic preparation for the identification of the serological groups of fi-hemolytic streptococci consisting essentially of a carrier having:

(A) A first area impregnated with glycerol and an indicator system which is capable of indicating pH changes between about 9 to about 5;

(B) A second area comprising a hydrophobic carrier separating the first area from the third area.

(C) A third area impregnated with salicin and a pH indicator system capable of indicating pH changes from about 9 to 5.

(D) A fourth area comprising a hydrophobic carrier separating the third area from the fifth area.

(E) A fifth area impregnated with esculin and a member selected from the group consisting of a ferric salt, an aluminum salt and a lead salt.

(F) A sixth area comprising a hydrophobic barrier.

2. A diagnostic preparation according to claim 1 wherein said indicator system in the glycerol area is a member selected from the group consisting of chlorophenol red, bromocresol purple, bromophenol red, bromothymol blue, neutral red, a-naphtholphthalein and a phenol red.

3. -A diagnostic preparation according to claim 1 wherein said indicator system in the salicin area is a member selected from the group consisting of chlorophenol red, bromocresol purple, bromophenol red, bromothymol blue, neutral red, ot-naphtholphthalein and phenol red.

4. A diagnostic preparation according to claim 1 wherein the glycerol area and the salicin area also contain nutrient media.

5. A diagnostic preparation according to claim 1 wherein said ferric salt is a member selected from the group consisting of ferric chloride, ferric ammonium citrate, ferric nitrate and ferric citrate.

6. A diagnostic preparation according to claim 1 wherein said aluminum salt is aluminum ammonium citrate.

7. A diagnostic preparation according to claim 1 wherein said lead salt is a member selected from a group consisting of lead acetate and lead nitrate.

8. A process for the serological identification of fl-hemolytic streptococci by inoculating organisms onto the glycerol area, onto the salicin area and onto the esculin area of the diagnostic preparation of claim 1; incubating the resulting system at 37 C. for about 2 to 4 hours and observing these areas for color change.

(References on following page) Referencs Cited Weaver et al., Am. J. Clin. Path. 49 (4):494499 UNITED STATES PATENTS (1968)- 2,904,474 9/1959 195103-5 iR X A. LOUIS MONAC-ELL, Primary Examiner 3,597,321 8/1971 Kronlsh 19s 103.s R 5 MD'HENSLEY, AssistamEXaminer OTHER REFERENCES US. Cl- X' R.

Bergeys Manual of Determinative Bacterology, 7th 23 253 TP ed., pp. 510-529 (1957). 

